SOLUTION X-RAY SCATTERING STUDIES ON ALLOSTERIC TRANSITION OF E COLI ASPARTATE

大肠杆菌天门冬氨酸变构转变的溶液X射线散射研究

• 批准号: 7954205
• 负责人: HIROTSUGU TSURUTA
• 金额: $ 0.25万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954205
• 关键词: Active Sites; Amino Acids; Anabolism; Aspartate; Binding; Binding Sites; Carbamoyl Transferases; Cells; Computer Retrieval of Information on Scientific Projects Database; Crystallography; Data; Enzymes; Equilibrium; Escherichia coli; Exhibits; Feedback; Funding; Grant; Hydrogen Bonding; Institution; Ligand Binding; Link; N phosphonoacetyl L aspartate; Pathway interactions; Physiological; Pyrimidine; Pyrimidines; Regulation; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Role; Sodium Chloride; Solutions; Source; Structure; Techniques; Time; United States National Institutes of Health; analog; mutant; structural biology; synchrotron radiation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. E. coli aspartate transcarbamoylase is a classical example of allosteric enzyme, possessing homotropic and heterotropic cooperativities, which have a fundamental role in feedback regulation in the pyrimidine biosynthesis pathway. We combine x-ray crystallography and solution x-ray solution techniques to study a large scale quaternary structure change associated with the allosteric transition. High resolution structures obtained by crystallography are sometimes biased by the physical constraints of the unit cell, and solution scattering is used to evaluate crystal structures as well as to obtain true physiological structures in solution. Solution x-ray scattering is frequently used to investigate time-course of structural change upon ligand binding. We have recently investigated several mutant versions of ATCase, all containing substitutions of critical amino acid residues within the active site by equilibrium and time-resolved solution x-ray scattering. Two mutant enzymes H134A and R167Q, remained in the T state after addition of a saturating concentration of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA), and two others, R229A and Q231L, were shifted only partly towards the R state. The mutant enzymes that did exhibit a shift towards the R state after addition of the natural substrates (S52A, K84A, Q231L and R296A) all had much slower T -> R transition rates than the wild-type enzyme. Most strikingly the T -> R transition rate for these mutant enzymes after addition of PALA was unchanged as compared to the wild-type enzyme. These data indicate that the loss of a single interaction in the binding site is not enough to eliminate the ability of the enzyme to undergo the T to R transition, unless the residue making that interaction is also involved in an interdomain R-state stabilizing salt link or forms hydrogen bonds with other active site residues. In addition, the data suggest different pathways for the allosteric transition after the binding of PALA as compared to the natural substrate

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 E.大肠杆菌天冬氨酸转氨甲酰酶是一种典型的变构酶，具有同向和异向协同作用，在嘧啶生物合成途径的反馈调节中起重要作用。我们结合联合收割机X射线晶体学和溶液X射线溶液技术来研究与变构转变相关的大规模四级结构变化。通过晶体学获得的高分辨率结构有时会受到晶胞物理约束的影响，溶液散射用于评估晶体结构以及获得溶液中真实的生理结构。溶液X射线散射经常用于研究配体结合后结构变化的时间过程。我们最近研究了几个突变版本的ATCase，所有包含的关键氨基酸残基的活性位点内的平衡和时间分辨的溶液X-射线散射的取代。两个突变酶H134 A和R167 Q，在加入饱和浓度的双底物类似物N-膦酰乙酰基-L-天冬氨酸（PALA）后保持在T状态，另外两个，R229 A和Q231 L，仅部分向R状态转移。在加入天然底物后确实表现出向R状态转变的突变体酶（S52 A、K84 A、Q231 L和R296 A）都具有比野生型酶慢得多的T -> R转变速率。 最引人注目的是，与野生型酶相比，这些突变酶在添加PALA后的T -> R转换速率没有变化。这些数据表明，在结合位点的单一相互作用的损失是不足以消除酶的能力，进行T到R的转变，除非残基的相互作用也参与了一个域间的R-状态稳定盐连接或形成氢键与其他活性位点的残基。 此外，数据表明与天然底物相比，PALA结合后的变构转变途径不同